氟喹诺酮类药物多残留酶联免疫检测方法的建立

Development of an ELISA Method for Multi-Residue Detecting of Fluoroquinolones

【目的】氟喹诺酮类药物(FQs)在畜牧业中广泛用于预防和治疗细菌性疾病,不合理使用导致在动物性食品中残留,严重危害消费者健康,其检测受到世界各国重视。制备抗多种FQs单克隆抗体(m Abs),建立间接竞争ELISA(ic ELISA)检测方法,为动物性食品中FQs多残留检测奠定基础。【方法】环丙沙星(CPFX)羧基上引入氨基丁酸后为半抗原(CPFX-A),质谱鉴定;分别用碳二亚胺法(DDC)和混合酸干法将半抗原与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原(CPFX-A-BSA)和包被原(CPFX-A-OVA),紫外和红外光谱鉴定是否偶联成功;CPFX-A-BSA免疫Balb/c小鼠,间接ELISA和ic ELISA方法测定多抗血清效价和敏感性,选取抗血清效价高和敏感性好的鼠用于细胞融合;NS0细胞和脾细胞按1﹕5比例在PEG-1500作用下融合,筛选抗FQs杂交瘤细胞株,并进行效价、亚型、敏感性和交叉反应率鉴定;体内诱生腹水法制备m Ab,液体石蜡作为诱导剂,每只鼠注射108个杂交瘤细胞;选敏感性和广谱特异性好的腹水m Ab,建立ic ELISA标准曲线,对ic ELISA检测方法优化,在鸡肉中添加10种FQs进行测定,将测定结果与HPLC法比较,用SPSS 17.0软件进行显著差异性分析。【结果】半抗原改造和人工抗原合成成功;3只鼠血清效价均达到1﹕1.28×104以上,2号鼠血清效价最高(1﹕2.56×104),且IC50值最低(12.92 ng·m L-1),选择2号鼠作为细胞融合用鼠;4次亚克隆筛选并鉴定后获得3株敏感广谱特异的杂交瘤细胞,分别命名为2H5、3D11、4F4,细胞上清效价分别为1﹕1 600、1﹕1 600和1﹕800,腹水效价分别为1﹕1.6×106、1﹕8.2×105和1﹕8.2×105;ic ELISA方法的包被原浓度为1μg·m L-1,5%猪阴性血清4℃包被过夜,单抗1﹕40 000稀释,山羊抗鼠酶标二抗(Ga MIg G-HRP)1﹕8 000稀释;反应温度均为37℃,标准品与单抗同时加入反应15 min,洗板后加二抗反应25 min;显色10 min,2H5细胞株腹水敏感性和广谱特异性最好,2H5的线性回归方程为y=-28.022x+56.219,R2=0.9 782,对10种FQs的IC50值分别为环丙沙星(CPFX)1.67ng·m L-1、诺氟沙星(NOR)1.82 ng·m L-1、培氟沙星(PEF)1.97 ng·m L-1、恩诺沙星(ENR)1.54 ng·m L-1、单诺沙星(DAN)2.79 ng·m L-1、洛美沙星(LOM)3.38 ng·m L-1、氧氟沙星(OFL)5.50 ng·m L-1、麻保沙星(MAR)4.40 ng·m L-1、沙拉沙星(SAR)11.76 ng·m L-1、二氟沙星(DIF)13.60 ng·m L-1,与10种FQs的交叉反应率为12.3%—108.4%,与非FQs交叉反应率均低于0.01%,对10种FQs的检测限(LODs)为0.09 ng·m L-1—0.64 ng·m L-1。ic ELISA法鸡肉中10种FQs的回收率为80.5%—91.8%,HPLC法的回收率为85.1%—95.7%,二者变异系数均低于10.0%;两种检测方法差异不显著(P>0.05)。【结论】该试验获得了高效价、敏感、广谱特异性的m Abs,建立的ic ELISA方法可用于同时检测鸡肉中10种FQs。

【 Objective 】 Fluoroquinolones（FQs） are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study was to produce class-specific monoclonal antibodies（m Abs） against fluoroquinolones（FQs）, establish competitive indirect enzyme linked immunesorbent assay（ic ELISA）, and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten（CPFX-A） and was proved by（＋） ESI-MS spectrum, which was conjugated to bovine serum albumin（BSA） as immunogen（CPFX-A-BSA） by the N,N＇-Dicyclohexylcarbodiimide（DDC） method, and to ovalbumin（OVA） as coating antigen（CPFX-A-OVA） by mixed anhydride method, respectively, which were then identified by infrared ray（IR） and ultraviolet（UV）. Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and ic ELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete m Ab against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 108 hybridoma cells in vivo, and ic ELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×104, in which the titer and IC50 of No.2 mouse were 1﹕2.56×104 and 12.92 ng·m L^-1. Three hybridoma cell lines named 2H5, 3D11, and 4F4 were screened after 4 times subclone, which titers were 1﹕1 600, 1﹕1 600, and 1﹕800 in supernatants and 1﹕1.6×10^6, 1﹕8.2×10^5, and 1﹕ 8.2×105 in ascites, respectively. The ic ELISA procedure was optimized at a concentration of CPFX- A-OVA for 1 μg·m L-1 at 4℃ package overnight by 5% negative serum of pig, monoclonal antibody and Ga MIg G-HRP were diluted 1 ﹕ 40 000 and 1 ﹕ 8 000, respectively. Under the reaction temperature of 37℃, reaction time of standard substance and monoclonal antibody was 15 min, and 25 min after adding Ga MIg G-HRP, also 10 min for termination reaction. Cell line named 2H5 showed a good sensitivity and class-specific toward 10 FQs, the linear regression equation was y=-28.022x＋56.219,R2=0.9 782,with an IC50 value of 1.67 ng.mL^-1 for ciprofloxacin, 1.82 ng.mL^-1 for norfloxacin, 1.97 ng.mL^-1 for pefloxacin, 1.54 ng.mL^-1 for enrofloxacin, 2.79 ng.mL^-1 for danofloxacin, 3.38 ng.mL^-1 for lomefloxacin, 5.50 ng.mL^-1 for ofloxacin, 4.40 ng.mL^-1 for marbofloxacin, 11.76 ng.mL^-1 for sarafloxacin, 13.60 ng.mL^-1 for difloxacin, and the lowest detectable limits（LODs） of 0.09ng.mL^-1-0.64 ng.mL^-1 and cross-reactivity（CR） of 12.3%-108.4%, no cross-reactivity to other compounds was found. The recovery ranges of 10 FQs spiked in chicken using ic ELISA were 80.5%-91.8%, 85.1%-95.7% for HPLC, both of the coefficient variations（CVs） were below 10.0%, and no significant difference（P0.05） was observed.【Conclusion】The high-sensitivity and class-specific m Ab against FQs was prepared, which laid a solid foundation for FQs multi-residue detection.